1. Field of the Invention
The invention relates to primer-specific and mispair extension assay for identifying gene variations, such as in different genotypes or subtypes of a given genotype.
2. State of the Art
Current genotyping systems are technically complex, time-consuming and error-prone in the detection of a single nucleotide variation and low level heterozygotes.
Despite current genotyping systems, such as restriction fragment length polymorphism analysis (RFLP), hybridization for example, line probe assay (xe2x80x9cLiPAxe2x80x9d), selective DNA amplification by PCR-type-specific primers (Okamoto, H., et al., J. Gen. Virol. 73: 673, 678, 1992) and direct DNA sequencing having been useful in general, some technical problems still remain and limit their applications.
Development of viral resistance to antiviral drugs used for treatment of HIV-1 infection is an important cause of treatment failure (Coffin J M., Sciences, 267: 483-489, 1995). In addition, drug-resistant mutations can give rise to selective cross-resistance to other antiviral drugs which has limited the options available for alternative antiviral regimens (Cohen O J, and Fauci A S., N. Engl. J. Med., 339: 341-243, 1998). Thus, genotypic drug resistance testing plays an important role in selecting an initial antiviral regimen and changing therapy with alternate regimens as the need arises. However, current genotyping assays, including direct DNA sequencing, yield information on only the predominant viral quasispecies due to the inability of these assays to detect low levels of viral variants (Gxc3xcnthard, H. F., et al., AIDS Res. Hum. Retroviruses, 14: 869-876, 1998). An ABI automated sequencer was able to detect mutants at levels of 10-50% in an artificially mixed sample only when the mixes were analyzed by editing the sequences manually. Hybridization-based high-density oligonucleotide arrays (GeneChip) (Deeks S G, Abrams D I., Lancet, 349: 1489-1490, 1997) were less sensitive than the ABI and were able to detect mutations at levels of 25-75% in the mix (Gxc3xcnthard, H. F., et al, AIDS Res. Hum. Retroviruses, 14: 869-876, 1998). Similar results were obtained using an automated DNA sequencer (Visible Genetics) (Hu Y W, et al., Reliable detection of mixed HCV genotype infections using a novel genotyping assay. 5th International Meeting on Viral Hepatitis C Virus and Related Viruses, Molecular Virology and Pathogenesis, Venezia, Italy, 1998). Line Probe Assay (xe2x80x9cLiPAxe2x80x9d), which uses reverse hybridization technology, is relatively rapid and could detect mutants at levels as low as 5% (Stuyver L, et al., Antimicrob. Agents Chemother., 41: 284-291, 1997), but again, may not be suitable for detection of mixed genotypes because it may not give results due to nearby polymorphisms that impair hybridization (Stuyver L, et al., Antimicrob. Agents Chemother., 41: 284-291, 1997). Moreover, in 40% of the samples tested, LiPA failed to yield correct results for some of the drug-resistant mutations (Puchhammer-Stockl E, et al, J. Med. Virol., 57: 283-289, 1999). Population based sequencing, for example, cloning and sequencing, is the gold standard method for detection of minor drug-resistant mutants. Unfortunately, it is impractical for clinics and large cohort studies.
For most indirect DNA sequencing genotyping systems, a common weakness is that they are not as accurate as direct DNA sequencing analysis, particularly for detection of a single nucleotide mutation or variation, resulting in considerable instances of errors or inconsistent results (Andonov, A., et al., J. Clin. Microbiol. 32: 2031-2034, 1994; Tuveri, R., et al., Journal of Medical Virology 51: 36-41, 1997; Okamoto, H., et al., J. Virol. Methods 57: 31 45 002-16, 1996). Although direct DNA sequencing is the most reliable method for genotyping, this is not practical for large cohort studies.
For example, International Publication No. WO 91/13075 describes a method for detecting variable nucleotides based on primer extension and incorporation of detectable nucleoside triphosphate using T7 polymerase for extending the primer. However, T7 polymerase does not have the proofreading activity and the 3xe2x80x2-5xe2x80x2 exonuclease activity of pfu results in false positive or false negative reactions. Moreover, the method uses ddNTPs for terminating extension.
Another major limitation of all current genotyping systems, including direct DNA sequencing, is that they cannot reliably detect low levels of heterozygotes (Tuveri, R., et al, Journal of Medical Virology 51: 36-41, 1997; Lau, J. Y., et al., J. Infect. Dis. 171: 281-289, 1995; Forns, X., et al., J. Clin. Microbiology. 34-10: 2516-2521, 1996) or mixed genotype infections (Tuveri, R., et al., Journal of Medical Virology 51: 36-41, 1997; Lau, J. Y., et al., J. Infect. Dis. 171: 281-289, 1995; Forns, X., et al., J. Clin. Microbiology. 34-10: 2516-2521, 1996).
International Publication No. WO 96/30545 discloses a method for simultaneously analyzing a genetic mutation and a corresponding wild-type sequence within a sample. The method utilizes ddNTPs for terminating primer extension. However, the use of ddNTPs increases the background of the assay, reducing its sensitivity and capacity to detect low levels of heterozygotes, similarly as in WO 91/13075.
HCV was recognized as the major etiologic agent of blood borne non-A, non-B hepatitis soon after the virus was identified in 1989. As an RNA virus, HCV shows great genetic variability, resulting in the existence of types, subtypes and quasispecies. At present, 11 types and at least 50 subtypes have been described. However, types 1a, 1b, 2a, 2b and 3a have been found to be generally the most prevalent (Simmonds, P., Hepatology 21: 570-582, 1995). Subtype 1b is the most common genotype found in Japan (Okamoto, H., et al., J. Gen. Virol 73: 673, 678, 1992) and European countries while subtypes 1a and 1b are the most common genotypes in the United States (Lau, J. Y., et al., J. Infect. Dis. 171: 281-289, 1995) and Canada, (Andonov, A., et al., J. Clin. Microbiol. 32: 2031-2034, 1994). Viruses of various genotypes contain different antigenic properties, which have potentially important consequences for the development of a vaccine and for antibody screening tests. Also, the disease severity and response to interferon may be influenced by the virus types and subtypes (Simmonds, Hepatology 21: 570-582, 1995). Subtype 1b was reported to be associated with a high severity of the disease and low response to interferon (Simmonds, Hepatology 21: 570-582, 1995). It is apparent that a rapid, simple, accurate and inexpensive genotyping method is urgently needed.
Amplification refractory mutation system (xe2x80x9cARMSxe2x80x9d) (Newton, C. R., et al., Nucl. Acids Res. 17: 2503-2516), improved the methods used in the prior art for typing the five most common genotypes (Pistello, M., et al., J. Clin. Microbiol. 32: 232-234, 1994). ARMS was developed for PCR detection of any point mutation in DNA using Taq DNA polymerase (Newton, C. R., et al., Nucl. Acids Res. 17: 2503-2516) and is based on the principle that oligonucleotides with a mismatched 3xe2x80x2-residue would not function as primers in PCR under controlled conditions. In some cases, however, the specificity of ARMS was insufficient to give a correct diagnosis. The problem with nonspecific reactivities still remains with the type-specific primer PCR method for HCV genotyping, even with the improvement using ARMS.
It is apparent that the major cause of the nonspecific reactivities found in these assays is related to the use of Taq DNA polymerase due to its lack of 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity. This inaccuracy results in base substitutions, transitions, tranversions, frame shifts or deletion mutations during DNA synthesis. Consequently, mispairs can be frequently formed, and Taq polymerase would be able to continue synthesizing DNA by addition of the next correct nucleotide on the template (Lau, J. Y., et al., J. Infect Dis. 171: 281-289, 1995). Even after reaching the end of the template, several more nucleotides can be added to the extended primer because most DNA polymerases, including Taq and retroviral reverse transcriptase (RT), have a nontemplate-dependent DNA synthesis activity, for example, terminal deoxynucleotide transferase activity (Clark, J. M., Nucleic Acids Res. 16: 9677; and Patel, P. H., et al., Proc. Natl. Acad. Sci. U.S.A. 91: 549-553). Therefore, the nonspecific reaction cannot be avoided with either ARMS or the methods based on ARMS using Taq DNA polymerase.
Two thermostable DNA, polymerases pfu (Pyrococcus furiosus) (Lundberg, K. S., et al., Pyrococcus furiousus. Gene 108: 1-6, 1991), and TLI/Vent (Thermocococcus litoralis), (Neuner, A., et al., Iarch. Microbiol., 153: 205-207), exhibit 3xe2x80x2xe2x86x925xe2x80x2 proofreading exonuclease activity. This ensures a high degree of amplification fidelity during DNA polymerization. Unlike Vent, the pfu 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity peaks sharply at its optimal polymerization temperature (75xc2x0 C. to 80xc2x0 C.), minimizing undesirable primer-degradation activity (Lundberg, K. S., et al., Gene 108: 1-6, 1991). Pfu DNA polymerase also does not exhibit terminal deoxynucleotidyl-transferase (TDT) activity, which was reported to be involved in the high mutation rate of DNA during DNA polymerization.
It would be highly desirable to be provided with a simple assay or method to overcome many of these limitations of current DNA genotyping systems.
It would be highly desirable to be provided with genotypic assays that possess greater accuracy and sensitivity for detection of minor drug-resistant subpopulations of HIV-1 in the early stages of resistance evolution.
One aim of the present invention is to provide a novel primer-specific and mispair extension assay (PSMEA) for determining genotypes and subtypes.
Another aim of the present invention is to provide a primer-specific and mispair extension assay (PSMEA) for detecting nucleotide variations in any known gene sequence using pfu DNA polymerase in the presence of an incomplete set of dNTPs and only a single primer.
Another aim of the present invention is to provide a tool for reliable detection of mixed genotype infection.
Another aim of the present invention is to provide a sensitive tool for detecting low levels of drug-resistant mutants in patients being treated with antiviral drugs.
Another aim of the present invention is to provide a tool for accurate genotyping.
In accordance with the present invention, there is provided a primer-specific and mispair extension assay for determining genotype. The assay includes:
a) extending a nucleic acid sequence from a patient sample with a polymerase, more preferably a pfu DNA polymerase, using a primer-specific for a genotype to be determined and an incomplete set of dNTPs, under suitable conditions for obtaining extension products of the primer, wherein at least one of the primer or the dNTPs is labeled;
b) characterizing the extension products; and
c) comparing the extension products with known nucleic acid sequences of various genotypes for determining the genotype of the nucleic acid sequence extended.
Preferably, characterizing the extension products includes separating the extension products by size. More preferably, characterizing the extension products further includes sequencing the extension products after separating the extension products by size.
Alternatively, the assay may further include amplifying the nucleic acid sequence before extending the nucleic acid sequence from a patient sample.
Preferably, the incomplete set of dNTPs contains two or three different types of nucleotides.
In a preferred embodiment, the primer is labeled with a radioactive label or a fluorescent label.
In another embodiment, one of the dNTPs is labeled with a radioactive label or a fluorescent label.
In accordance with the present invention, there is provided a novel genotyping system, primer-specific and mispair extension analysis (PSMEA) preferably using the unique 3xe2x80x2 to 5xe2x80x2 exonuclease proofreading properties of pfu DNA polymerase with an incomplete set of dNTPs and only a single primer.
Also in accordance with one embodiment of the present invention there is provided a primer-specific and mispair extension assay for determining genotype and detecting low level mixed genotype injections or heterozygotes. The assay includes:
a) extending a DNA sequence, amplified from a patient sample, with pfu DNA polymerase using a primer-specific for a genotype to be determined and an incomplete set of dNTPs, under suitable conditions for extending the primer, wherein at least one of the primer or one of the dNTPs is labeled;
b) separating the extended DNA sequences obtained in step a);
c) detecting the separated extended DNA sequences; and
d) comparing the extended DNA sequences with known DNA sequences of various genotypes for determining the genotype of the DNA sequences extended.
The primer may be end-labeled with a label or one of the dNTPs can be labeled with a label. The label can be a radioactive or fluorescent label.
Preferably, the steps a), b), c), and d) as described above are automated.